These investigations will focus on the potential interrelationship between age-dependent structural alterations in the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and basic mechanisms of nitric oxide (NO) action in human cells. GAPDH is a unique in vivo target of nitric oxide which oxidatively induces its covalent modification by NAD+. GAPDH also is involved in neuronal cell apoptosis and is associated with the cellular phenotype of a series of age-related neurodegenerative -disorders, including Alzheimer's Disease and Huntington's Disease. Recent evidence indicates that GAPDH is involved in a number of membrane, cytoplasmic and nuclear cell functions apart from its classical role in glycolysis. The goal of this pilot proposal is to examine the relationship between age- related GAPDH structural alterations and NO-induced oxidative stress. Early and late passage human cells will be used as an experimental paradigm. Sodium nitroprusside will be used to generate NO in vivo. Age-related structural changes in NO-induced NAD modified GAPDH will be defined using SDS-PAGE densitometric analysis and anti- human GAPDH monoclonal antibodies developed in this laboratory. Structure-function studies will include in vivo analysis of oligomeric GAPDH structure, changes in its subcellular localization as well as kinetic analysis of NAD modified GAPDH dissociation and reassociation as a function of NO exposure. In this manner, initial information may be gathered to indicate whether there is sufficient evidence to justify detailed studies to examine the interrelationship between age-dependent changes in an important nervous system protein, oxidative stress and the role of nitric oxide in the biology of aging.